Project Summary Cyanobacterial bloom events are shaped by environmental cues that contribute to harmful algal bloom (cHAB) composition and toxigenicity. Indeed, in Lake Erie, two distinctly different cyanobacterial genera, Microcystis and Planktothrix, produce microcystin, a potent hepatotoxin. Whereas both cyanobacteria bloom in waters affected by nutrients delivered by agricultural runoff, Microcystis typically blooms in offshore waters, and Planktothrix dominates nearshore embayments. Prior work has suggested a role of climate-change induced temperature increases in promoting growth of Microcystis, and that Planktothrix persists in waters affected by prolonged nitrogen depletion. Given the different characteristics of these two organisms, limiting exposure to microcystin may require different management strategies to protect the water supply. Our overarching aim in this project is to address the extent to which N availability and temperature play in the development, toxigenicity and persistence of cHAB taxa in Lake Erie and other bloom-affected freshwaters. Informed by baseline data from metatranscriptomic surveys, we will design microcosm and chemostat experiments aimed at understanding competition between the two Lake Erie cHAB genera and factors contributing to bloom toxicity. Specifically, we will test three hypotheses in this project listed below: 1) Nitrogen availability controls cHAB community structure. Microcosms of mixed Microcystis and Planktothrix bloom communities will be exposed to different regimes of N availability and temperature to determine the degree to which each contribute to the dominance of one bloom-former over another. 2) Microcystin production is dependent on temperature and N speciation. Chemostat studies of Microcystis and Planktothrix cultures at control and warmer temperature will reveal how different chemical forms of N (nitrate vs. urea) promote growth and toxin production. 3) The proliferation of cHAB species is promoted by the activity of the limnetic microbiome. Metatransciptomic analysis of bloom communities will provide an assessment of the activities of the entire microbial consortium in cHAB events in order to determine what organisms and pathways assist in cHAB development, persistence and decline. A second aim of this project is to generate a comprehensive Lake Erie environmental `omics data set that can be shared and utilized by the integrated research projects within the Great Lakes Center for Fresh Waters and Human Health.